Peptide-based compounds

ABSTRACT

The invention relates to new peptide-based compounds for use as diagnostic imaging agents or as therapeutic agents wherein the agents comprise targeting vectors which bind to integrin receptors.

This application is a continuation of U.S. application Ser. No.12/423,953 filed Apr. 15, 2009, now U.S. Pat. No. 7,994,134, which is adivision of U.S. application Ser. No. 10/753,729 filed Jan. 8, 2004, nowU.S. Pat. No. 7,521,419, which is a continuation application ofinternational application number PCT/NO02/00250 filed Jul. 8, 2002,which claims priority to Great Britain application number 0116815.2filed Jul. 10, 2001 and to Norwegian application number 20014954 filedOct. 11, 2001, the entire disclosure of which is hereby incorporated byreference.

FIELD OF INVENTION

The present invention relates to new peptide-based compounds and theiruse in therapeutically effective treatments as well as for diagnosticimaging techniques. More specifically the invention relates to the useof such peptide-based compounds as targeting vectors that bind toreceptors associated with angiogenesis, in particular integrinreceptors, e.g. the αvβ3 integrin receptor. Such contrast agents maythus be used for diagnosis of for example malignant diseases, heartdiseases, endometriosis, inflammation-related diseases, rheumatoidarthritis and Kaposi's sarcoma. Moreover such agents may be used intherapeutic treatment of these diseases.

BACKGROUND OF INVENTION

New blood vessels can be formed by two different mechanisms:vasculogenesis or angiogenesis. Angiogenesis is the formation of newblood vessels by branching from existing vessels. The primary stimulusfor this process may be inadequate supply of nutrients and oxygen(hypoxia) to cells in a tissue. The cells may respond by secretingangiogenic factors, of which there are many; one example, which isfrequently referred to, is vascular endothelial growth factor (VEGF).These factors initiate the secretion of proteolytic enzymes that breakdown the proteins of the basement membrane, as well as inhibitors thatlimit the action of these potentially harmful enzymes. The otherprominent effect of angiogenic factors is to cause endothelial cells tomigrate and divide. Endothelial cells that are attached to the basementmembrane, which forms a continuous sheet around blood vessels on thecontralumenal side, do not undergo mitosis. The combined effect of lossof attachment and signals from the receptors for angiogenic factors isto cause the endothelial cells to move, multiply, and rearrangethemselves, and finally to synthesise a basement membrane around the newvessels.

Angiogenesis is prominent in the growth and remodelling of tissues,including wound healing and inflammatory processes. Tumors must initiateangiogenesis when they reach millimeter size in order to keep up theirrate of growth. Angiogenesis is accompanied by characteristic changes inendothelial cells and their environment. The surface of these cells isremodelled in preparation for migration, and cryptic structures areexposed where the basement membrane is degraded, in addition to thevariety of proteins which are involved in effecting and controllingproteolysis. In the case of tumours, the resulting network of bloodvessels is usually disorganised, with the formation of sharp kinks andalso arteriovenous shunts. Inhibition of angiogenesis is also consideredto be a promising strategy for antitumour therapy. The transformationsaccompanying angiogenesis are also very promising for diagnosis, anobvious example being malignant disease, but the concept also showsgreat promise in inflammation and a variety of inflammation-relateddiseases, including atherosclerosis, the macrophages of earlyatherosclerotic lesions being potential sources of angiogenic factors.These factors are also involved in re-vascularisation of infarcted partsof the myocardium, which occurs if a stenosis is released within a shorttime.

Further examples of undesired conditions that are associated withneovascularization or angiogenesis, the development or proliferation ofnew blood vessels are shown below. Reference is also made in this regardto WO 98/47541.

Diseases and indications associated with angiogenesis are e.g. differentforms of cancer and metastasis, e.g. breast, skin, colorectal,pancreatic, prostate, lung or ovarian cancer.

Other diseases and indications are inflammation (e.g. chronic),atherosclerosis, rheumatoid arthritis and gingivitis.

Further diseases and indications associated with angiogenesis arearteriovenous alformations, astrocytomas, choriocarcinomas,glioblastomas, gliomas, hemangiomas (childhood, capillary), hepatomas,hyperplastic endometrium, ischemic myocardium, endometriosis, Kaposisarcoma, macular degeneration, melanoma, neuroblastomas, occludingperipheral artery disease, osteoarthritis, psoriasis, retinopathy(diabetic, proliferative), scleroderma, seminomas and ulcerativecolitis.

Angiogenesis involves receptors that are unique to endothelial cells andsurrounding tissues. These markers include growth factor receptors suchas VEGF and the Integrin family of receptors. Immunohistochemicalstudies have demonstrated that a variety of integrins perhaps mostimportantly the α_(v) class are expressed on the apical surface of bloodvessels [Conforti, G., et al. (1992) Blood 80: 37-446] and are availablefor targeting by circulating ligands [Pasqualini, R., et al. (1997)Nature Biotechnology 15: 542-546]. The α5β1 is also an importantintegrin in promoting the assembly of fibronectin matrix and initiatingcell attachment to fibronectin. It also plays a crucial role in cellmigration [Bauer, J. S., (1992) J. Cell Biol. 116: 477-487] as well astumour invasion and metastasis [Gehlsen, K. R., (1988) J. Cell Biol.106: 925-930].

The integrin αvβ3 is one of the receptors that is known to be associatedwith angiogenesis. Stimulated endothelial cells appear to rely on thisreceptor for survival during a critical period of the angiogeneicprocess, as antagonists of the αvβ3 integrin receptor/ligand interactioninduce apoptosis and inhibit blood vessel growth.

Integrins are heterodimeric molecules in which the α- and β-subunitspenetrate the cell-membrane lipid bilayer. The α-subunit has four Ca²⁺domains on its extracellular chain, and the β-subunit has a number ofextracellular cysteine-rich domains.

Many ligands (eg. fibronectin) involved in cell adhesion contain thetripeptide sequence arginine-glycine-aspartic acid (RGD). The RGDsequence appears to act as a primary recognition site between theligands presenting this sequence and receptors on the surface of cells.It is generally believed that secondary interactions between the ligandand receptor enhance the specificity of the interaction. These secondaryinteractions might take place between moieties of the ligand andreceptor that are immediately adjacent to the RGD sequence or at sitesthat are distant from the RGD sequence.

RGD peptides are known to bind to a range of integrin receptors and havethe potential to regulate a number of cellular events of significantapplication in the clinical setting. (Ruoslahti, J. Clin. Invest., 87:1-5 (1991)). Perhaps the most widely studied effect of RGD peptides andmimetics thereof relate to their use as anti-thrombotic agents wherethey target the platelet integrin GpIIbIIIa.

Inhibition of angiogenesis in tissues by administration of either anαvβ3 or αvβ5 antagonist has been described in for example WO 97/06791and WO 95/25543 using either antibodies or RGD containing peptides. EP578083 describes a series of mono-cyclic RGD containing peptides and WO90/14103 claims RGD-antibodies. Haubner et al. in the J. Nucl. Med.(1999); 40: 1061-1071 describe a new class of tracers for tumourtargeting based on monocyclic RGD containing peptides. Biodistributionstudies using whole-body autoradiographic imaging revealed however thatthe ¹²⁵I-labelled peptides had very fast blood clearance rates andpredominantly hepatobiliary excretion routes resulting in highbackground.

Cyclic RGD peptides containing multiple bridges have also been describedin WO 98/54347 and WO 95/14714. Peptides derived from in vivo biopanning(WO 97/10507) have been used for a variety of targeting applications.The sequence CDCRGDCFC (RGD-4C), has been used to target drugs such asdoxirubicin (WO 98/10795), nucleic acids and adenoviruses to cells (seeWO 99/40214, WO 99/39734, WO 98/54347, WO 98/54346, U.S. Pat. No.5,846,782). Peptides containing multiple cysteine residues do howeversuffer from the disadvantage that multiple disulphide isomers can occur.A peptide with 4 cysteine residues such as RGD-4C has the possibility offorming 3 different disulphide folded forms. The isomers will havevarying affinity for the integrin receptor as the RGD pharmacophore isforced into 3 different conformations.

Further examples of RGD comprising peptide-based compounds are found inPCT/NO01/00146 and PCT/NO01/00390, the content of which are incorporatedherein by reference.

The efficient targeting and imaging of integrin receptors associatedwith angiogenesis in vivo demands therefore a selective, high affinityRGD based vector that is chemically robust and stable. Furthermore, theroute of excretion is an important factor when designing imaging agentsin order to reduce problems with background. These stringent conditionsare met by the bicyclic structures described in the present invention.

SUMMARY OF THE INVENTION

In one aspect, the invention provides new peptide-based compound ofFormula I as defined in the claims. These compounds have affinity forintegrin receptors, e.g. affinity for the integrin αvβ3.

The present invention also provides a pharmaceutical compositioncomprising an effective amount (e.g. an amount effective for enhancingimage contrast in in vivo imaging) of a compound of general formula I ora salt thereof, together with one or more pharmaceutically acceptableadjuvants, excipients or diluents.

The invention further provides a pharmaceutical composition fortreatment of a disease comprising an effective amount of a compound ofgeneral formula I, or an acid addition salt thereof, together with oneor more pharmaceutically acceptable adjuvants, excipients or diluents.

In a further embodiment of this invention, the use of radioisotopes ofiodine or fluorine is specifically contemplated. These species can beused in therapeutic and diagnostic imaging applications. While, at thesame time, a metal attached to a chelating agent on the samepeptide-linker can also be used in either therapeutic or diagnosticimaging applications.

Use of the compounds of formula I in the manufacture of therapeuticcompositions (medicament) and in methods of therapeutic or prophylactictreatment, preferably treatment of cancer, of the human or animal bodyare thus considered to represent further aspects of the invention.

Viewed from a further aspect the invention provides the use of acompound of formula I for the manufacture of a contrast medium for usein a method of diagnosis involving administration of said contrastmedium to a human or animal body and generation of an image of at leastpart of said body.

Viewed from a still further aspect the invention provides a method ofgenerating an image of a human or animal body involving administering acontrast agent to said body, e.g. into the vascular system andgenerating an image of at least a part of said body to which saidcontrast agent has distributed using scintigraphy, PET or SPECTmodalities, wherein as said contrast agent is used an agent of formulaI.

Viewed from a still further aspect the invention provides a method ofgenerating enhanced images of a human or animal body previouslyadministered with a contrast agent composition comprising a compound asdefined by formula I, which method comprises generating an image of atleast part of said body.

Viewed from a further aspect the invention provides a method ofmonitoring the effect of treatment of a human or animal body with a drugto combat a condition associated with cancer, preferably angiogenesis,e.g. a cytotoxic agent, said method involving administering to said bodyan agent of formula I and detecting the uptake of said agent by cellreceptors, preferably endothelial cell receptors and in particular αvβ3receptors, said administration and detection optionally but preferablybeing effected repeatedly, e.g. before, during and after treatment withsaid drug.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

Viewed from one aspect the invention provides new peptide-basedcompounds of Formula I as defined in the claims. These compounds haveaffinity for integrin receptors, e.g. affinity for the integrin αvβ3.

The compounds of Formula I comprise at least two bridges, wherein onebridge forms a disulphide bond and the second bridge comprises athioether (sulphide) bond and wherein the bridges fold the peptidemoiety into a ‘nested’ configuration.

The compounds of the current invention thus have a maximum of onedisulphide bridge per molecule moiety. Compounds defined by the presentinvention are surprisingly stable in vivo and under the conditionsemployed during labelling, e.g. during labelling with technetium.

These new compounds may be used in therapeutically effective treatmentsas well as for imaging purposes.

The new peptide-based compounds described in the present invention aredefined by Formula I:

or physiologically acceptable salts thereofwherein

-   -   G represents glycine, and    -   D represents aspartic acid, and    -   R₁ represents —(CH₂)_(n)— or —(CH₂)_(n)—C₆H₄—, preferably R₁        represents —(CH₂)—, and    -   n represents a positive integer between 1 and 10, and    -   h represents a positive integer 1 or 2, and    -   X₁ represents an amino acid residue wherein said amino acid        possesses a functional side-chain such as an acid or amine        preferentially aspartic or glutamic acid, lysine, homolysine,        diaminoalcylic acid or diaminopropionic acid,    -   X₂ and X₄ represent independently an amino acid residue capable        of forming a disulphide bond, preferably a cysteine or a        homocysteine residue, and    -   X₃ represents arginine, N-methylarginine or an arginine mimetic,        preferably an arginine, and    -   X₅ represents a hydrophobic amino acid or derivatives thereof,        preferably a tyrosine, a phenylalanine, a 3-iodo-tyrosine or a        naphthylalanine residue, and more preferably a phenylalanine or        a 3-iodo-tyrosine residue, and    -   X₆ represents a thiol-containing amino acid residue, preferably        a cysteine or a homocysteine residue, and    -   X₇ is absent or represents a homogeneous biomodifier moiety        preferably based on a monodisperse PEG building block comprising        1 to 10 units of said building block, said biomodifier having        the function of modifying the pharmacokinetics and blood        clearance rates of the said agents.

In addition X₇ may also represent 1 to 10 amino acid residues preferablyglycine, lysine, aspartic acid or serine. In a preferred embodiment ofthis invention X₇ represents a biomodifier unit comprised ofpolymerisation of the monodisperse PEG-like structure,17-amino-5-oxo-6-aza-3,9,12,15-tetraoxaheptadecanoic acid of Formula II,

-   -   wherein n equals an integer from 1 to 10 and where the        C-terminal unit is an amide moiety.    -   W₁ is absent or represents a spacer moiety and is preferentially        derived from glutaric and/or succinic acid and/or a        polyethyleneglycol based unit and/or a unit of Formula II

-   -   Z₁ is an antineoplastic agent, a chelating agent or a reporter        moiety that can be represented by a chelating agent of Formula        III

where:each R¹, R², R³ and R⁴ is independently an R group;each R group is independently H or C₁₋₁₀ alkyl, C₃₋₁₀ alkylaryl, C₂₋₁₀alkoxyalkyl, C₁₋₁₀ hydroxyalkyl, C₁₋₁₀ alkylamine, C₁₋₁₀ fluoroalkyl, or2 or more R groups, together with the atoms to which they are attachedform a carbocyclic, heterocyclic, saturated or unsaturated ring,or can represent a chelating agent given by formulas a, b, c and d.

A preferred example of a chelating agent is represented by formula e.

Conjugates comprising chelating agents of Formula III can beradiolabelled to give good radiochemical purity, RCP, at roomtemperature, under aqueous conditions at near neutral pH. The risk ofopening the disulphide bridges of the peptide component at roomtemperature is less than at an elevated temperature. A further advantageof radiolabelling the conjugates at room temperature is a simplifiedprocedure in a hospital pharmacy.

The role of the spacer moiety W₁ is to distance the relatively bulkychelating agent from the active site of the peptide component. Thespacer moiety W₁ is also applicable to distance a bulky antineoplasticagent from the active site of the peptide.

It is found that the biomodifier, X₇, modifies the pharmacokinetics andblood clearance rates of the compounds. The biomodifier effects lessuptake of the compounds in tissue i.e. muscle, liver etc. thus giving abetter diagnostic image due to less background interference. Thesecretion is mainly through the kidneys due to a further advantage ofthe biomodifier.

However the compounds defined in Formula I may also comprise chelatingagents, Z1, as defined in Table I.

In some aspects of the invention, Z₁ comprises a reporter moiety wheresaid reporter moiety comprises a radionuclide. Further definitions ofchelating agents are listed in the following Table I.

TABLE I Class of ligand Structure Definitions Amine- oxime

Y1-8 and be H, alkyl, aryl or combinations thereof and Y4 or Y5 containsa suitable functionality such that it can be conjugated to the peptidevector- e.g. preferably alkylamine, alkysulphide, alkoxy, alkylcarboxylate, arylamine, aryl sulphide or α-haloacetyl X = C or N when m′= n′ = 1 X = N when m′ = n′ = 2 MAG3 type

P = protecting group (preferably. benozyl, acetyl, EOE); Y1, Y2 containsa suitable functionality such that it can be conjugated to the peptidevector; preferably H (MAG3), or the side chain of any amino acid, ineither L or D form. G4 type ligands

Y1, Y2, Y3-contains a suitable functionality such that it can beconjugated to the peptide vector; preferably H, or the side chain of anyamino acid, in either L or D form. Tetra- amine ligands

Y1-Y-6 can be H, alkyl, aryl or combinations thereof where the Y1-6groups contain one or more functional moieties such that the chelate canbe con- jugated to the vector- e.g. preferably alkylamine,alkylsulphide, alkoxy, alkyl carboxylate, arylamine, aryl sulphide orα-haloacetyl Cylam type ligands

Y1-5 can be H, alkyl, aryl or combinations thereof and where Y1-5 groupscontain one or more functional moieties such that the chelate can becon- jugated to the vector- e.g. preferably alkylamine, alkylsulphide,alkoxy, alkyl carboxylate, arylamine, aryl sulphide or α-haloacetylDiamine- diphenol

Y1, Y2-H, alkyl, aryl and where Y1 or Y2 groups contains a functionalmoiety such that the chelate can be conjugated to the vector-e.g.preferably alkylamine, alkylsulfphide, alkoxy, alkyl carboxylate,arylamine, aryl sulphide or α-haloacetyl W = C, N m′ = n′ = 1 or 2 HYNIC

V = linker to vector or vector itself. Amide thiols

P = protecting group (preferably. benzoyl, acetyl, EOE); Y1-5 = H,alkyl, aryl; or Y3 is a L or D amino acid side-chain or glycine. and thecarb- oxylate may be used for conjugation to the vector via an amidebond. Alternatively the R₁₋₅ groups may contain additional functionalitysuch that the chelate can be conjugated to the vector- e.g. alkylamine,alkylsulphide, alkoxy, alkyl carboxylate, arylamine, aryl sulphide orα-haloacetyl.

In some aspects of the invention of Formula I the Z₁ moiety comprisesthe binding of a ¹⁸F isotope or an isotope of Cu, incorporation into theagent either as a prosthetic group or by substitution or additionreactions. The resulting compound may thus be used in Positron EmissionTomography (PET) Imaging.

In one aspect of the present invention of formula I Z₁ is represented byan antineoplastic agent. In this aspect the compound will target anangiogenic site associated with cancer and bring the antineoplasticagent to the diseased area.

The antineoplastic agent may be represented by cyclophosphamide,chloroambucil, busulphan, methotrexate, cytarabine, fluorouracil,vinblastine, paclitaxel, doxorubicin, daunorubicin, etoposide,teniposide, cisplatin, amsacrine, docetaxel, but a wide range of otherantineoplastic agents may also be used.

The peptide component of the conjugates described herein have preferablyno free amino- or carboxy-termini. This introduces into these compoundsa significant increase in resistance against enzymatic degradation andas a result they have an increased in vivo stability as compared to manyknown free peptides.

As used herein the term ‘amino acid’ refers in its broadest sense toproteogenic L-amino acids, D-amino acids, chemically modified aminoacids, N-methyl, Cα-methyl and amino acid side-chain mimetics andunnatural amino acids such as naphthylalanine. Any naturally occurringamino acid or mimetics of such natural occurring amino acids arepreferred.

Some preferred embodiments of the compounds of formula I are illustratedby compounds I-IV below:

In most cases, it is preferred that the amino acids in the peptide areall in the L-form. However, in some embodiments of the invention one,two, three or more of the amino acids in the peptide are preferably inthe D-form. The inclusion of such D-form amino acids can have asignificant effect on the serum stability of the compound.

According to the present invention, any of the amino acid residues asdefined in formula I may preferably represent a naturally occurringamino acid and independently in any of the D or L conformations.

Some of the compounds of the invention are high affinity RGD basedvectors. As used herein the term ‘high affinity RGD based vector’ refersto compounds that have a Ki of <10 nM and preferably <5 nM, in acompetitive binding assay for αvβ3 integrin and where the Ki value wasdetermined by competition with the known high affinity ligandechistatin. Methods for carrying out such competition assays are wellknown in the art.

The present invention also provides a pharmaceutical compositioncomprising an effective amount (e.g. an amount effective for enhancingimage contrast in in vivo imaging) of a compound of general formula I ora salt thereof, together with one or more pharmaceutically acceptableadjuvants, excipients or diluents.

The invention further provides a pharmaceutical composition fortreatment of a disease comprising an effective amount of a compound ofgeneral formula I, or an acid addition salt thereof, together with oneor more pharmaceutically acceptable adjuvants, excipients or diluents.

Other representative spacer (W₁) elements include structural-typepolysaccharides, storage-type polysaccharides, polyamino acids andmethyl and ethyl esters thereof, and polypeptides, oligosaccharides andoligonucleotides, which may or may not contain enzyme cleavage sites.

The reporter moieties (Z₁) in the contrast agents of the invention maybe any moiety capable of detection either directly or indirectly in anin vivo diagnostic imaging procedure. Preferably the contrast agentcomprises one reporter. Preferred are moieties which emit or may becaused to emit detectable radiation (e.g. by radioactive decay).

For MR imaging the reporter will either be a non zero nuclear spinisotope (such as ¹⁹F) or a material having unpaired electron spins andhence paramagnetic, superparamagnetic, ferrimagnetic or ferromagneticproperties; for light imaging the reporter will be a light scatterer(e.g. a coloured or uncoloured particle), a light absorber or a lightemitter; for magnetometric imaging the reporter will have detectablemagnetic properties; for electrical impedance imaging the reporter willaffect electrical impedance; and for scintigraphy, SPECT, PET, and thelike, the reporter will be a radionuclide.

Stated generally, the reporter may be (1) a chelatable metal orpolyatomic metal-containing ion (i.e. TcO, etc), where the metal is ahigh atomic number metal (e.g. atomic number greater than 37), aparamagentic species (e.g. a transition metal or lanthanide), or aradioactive isotope, (2) a covalently bound non-metal species which isan unpaired electron site (e.g. an oxygen or carbon in a persistant freeradical), a high atomic number non-metal, or a radioisotope, (3) apolyatomic cluster or crystal containing high atomic number atoms,displaying cooperative magnetic behaviour (e.g. superparamagnetism,ferrimagnetism or ferromagnetism) or containing radionuclides.

Examples of particular preferred reporter groups (Z₁) are described inmore detail below.

Chelated metal reporters are preferably chosen from the group below;^(H)Y, ^(99m)Tc, ¹¹¹In, ⁴⁷Sc, ⁶⁷Ga, ⁵¹Cr, ^(177m)Sn, ⁶⁷Cu, ¹⁶⁷Tm, ⁹⁷Ru,¹⁸⁸Re, ¹⁷⁷Lu, ¹⁹⁹Au, ²⁰³Pb and ¹⁴¹Ce.

The metal ions are desirably chelated by chelant groups on the linkermoiety. Further examples of suitable chelant groups are disclosed inU.S. Pat. No. 4,647,447, WO89/00557, U.S. Pat. No. 5,367,080, U.S. Pat.No. 5,364,613.

Methods for metallating any chelating agents present are within thelevel of skill in the art. Metals can be incorporated into a chelantmoiety by any one of three general methods: direct incorporation,template synthesis and/or transmetallation. Direct incorporation ispreferred.

Thus it is desirable that the metal ion be easily complexed to thechelating agent, for example, by merely exposing or mixing an aqueoussolution of the chelating agent-containing moiety with a metal salt inan aqueous solution preferably having a pH in the range of about 4 toabout 11. The salt can be any salt, but preferably the salt is a watersoluble salt of the metal such as a halogen salt, and more preferablysuch salts are selected so as not to interfere with the binding of themetal ion with the chelating agent. The chelating agent-containingmoiety is preferrably in aqueous solution at a pH of between about 5 andabout 9, more preferably between pH about 6 to about 8. The chelatingagent-containing moiety can be mixed with buffer salts such as citrate,carbonate, acetate, phosphate and borate to produce the optimum pH.Preferably, the buffer salts are selected so as not to interfere withthe subsequent binding of the metal ion to the chelating agent.

The following isotopes or isotope pairs can be used for both imaging andtherapy without having to change the radiolabeling methodology orchelator: ⁴⁷Sc₂₁; ¹⁴¹Ce₅₈; ¹⁸⁸Re₇₅; ¹⁷⁷Lu₇₁; ¹⁹⁹Au₇₉; ⁴⁷Sc₂₁; ¹³¹I₅₃;⁶⁷Cu₂₉; ¹³¹I₅₃ and ¹²³I₅₃; ¹⁸⁸Re₇₅ and ^(99m)Tc₄₃; ⁹⁰Y₃₉ and ⁸⁷Y₃₉;⁴⁷Sc₂₁ and ⁴⁴Sc₂₁; ⁹⁰Y₃₉ and ¹²³I₅₃; ¹⁴⁶Sm₆₂ and ¹⁵³Sm₆₂; and ⁹⁰Y₃₉ and¹¹¹In₄₉.

Preferred non-metal atomic reporters include radioisotopes such as ¹²³I,¹³¹I and ¹⁸F as well as non zero nuclear spin atoms such as ¹⁹F, andheavy atoms such as I.

In a further embodiment of this invention, the use of radioisotopes ofiodine or fluorine is specifically contemplated. For example, if thepeptide or linker is comprised of substituents that can be chemicallysubstituted by iodine or fluorine in a covalent bond forming reaction,such as, for example, substituents containing hydroxyphenyl orp-nitrobenzoyl functionality, such substituents can be labeled bymethods well known in the art with a radioisotope of iodine or fluorinerespectively. These species can be used in therapeutic and diagnosticimaging applications. While, at the same time, a metal attached to achelating agent on the same peptide-linker can also be used in eithertherapeutic or diagnostic imaging applications.

A preferred embodiment of the invention relates to a radiolabelled agentof general formula (I), particularly for use in tumour imaging.

The diagnostic agents of the invention may be administered to patientsfor imaging in amounts sufficient to yield the desired contrast with theparticular imaging technique. Where the reporter is a metal, generallydosages of from 0.001 to 5.0 mmoles of chelated imaging metal ion perkilogram of patient bodyweight are effective to achieve adequatecontrast enhancements. Where the reporter is a radionuclide, dosages of0.01 to 100 mCi, preferably 0.1 to 50 mCi will normally be sufficientper 70 kg bodyweight.

The dosage of the compounds of the invention for therapeutic use willdepend upon the condition being treated, but in general will be of theorder of from 1 pmol/kg to 1 mmol/kg bodyweight.

The compounds according to the invention may therefore be formulated foradministration using physiologically acceptable carriers or excipientsin a manner fully within the skill of the art. For example, thecompounds, optionally with the addition of pharmaceutically acceptableexcipients, may be suspended or dissolved in an aqueous medium, with theresulting solution or suspension then being sterilized.

The compounds of formula I may be therapeutically effective in thetreatment of disease states as well as detectable in in vivo imaging.Thus for example the vector on the reporter moieites may havetherapeutic efficacy, e.g. by virtue of the radiotherapeutic effect of aradionuclide reporter of the vector moiety.

Use of the compounds of formula I in the manufacture of therapeuticcompositions (medicament) and in methods of therapeutic or prophylactictreatment, preferably treatment of cancer, of the human or animal bodyare thus considered to represent further aspects of the invention.

Further examples of the reporters which may be used in the context ofthe current application are given on pages 63-66 and 70-86 of WO98/47541and the disclosures made on these pages are incorporated herein byreference in their entirety. It is hereby asserted that each and everyreporter or part thereof disclosed on the aforementioned pages isconsidered to be part of the description of the invention contained inthis application.

Viewed from a further aspect the invention provides the use of acompound of formula I for the manufacture of a contrast medium for usein a method of diagnosis involving administration of said contrastmedium to a human or animal body and generation of an image of at leastpart of said body.

Viewed from a still further aspect the invention provides a method ofgenerating an image of a human or animal body involving administering acontrast agent to said body, e.g. into the vascular system andgenerating an image of at least a part of said body to which saidcontrast agent has distributed using scintigraphy, PET or SPECTmodalities, wherein as said contrast agent is used an agent of formulaI.

Viewed from a still further aspect the invention provides a method ofgenerating enhanced images of a human or animal body previouslyadministered with a contrast agent composition comprising a compound asdefined by formula I, which method comprises generating an image of atleast part of said body.

Viewed from a further aspect the invention provides a method ofmonitoring the effect of treatment of a human or animal body with a drugto combat a condition associated with cancer, preferably angiogenesis,e.g. a cytotoxic agent, said method involving administering to said bodyan agent of formula I and detecting the uptake of said agent by cellreceptors, preferably endothelial cell receptors and in particular αvβ3receptors, said administration and detection optionally but preferablybeing effected repeatedly, e.g. before, during and after treatment withsaid drug.

The compounds of the present invention can be synthesised using all theknown methods of chemical synthesis but particularly useful is thesolid-phase methodology of Merrifield employing an automated peptidesynthesiser (J. Am. Chem. Soc., 85: 2149 (1964)). The peptides andpeptide chelates may be purified using high performance liquidchromatography (HPLC) and characterised by mass spectrometry andanalytical HPLC before testing in the in vitro screen.

The present invention will now be further illustrated by way of thefollowing non-limiting examples.

EXAMPLES Example 1 Synthesis of disulfide [Cys²⁻⁶] thioethercyclo[CH₂CO-Lys(cPn216-glutaryl)-Cys²-Arg-Gly-Asp-Cys⁶-Phe-Cys]-NH₂ (SEQID NO. 1)

1 a) Synthesis of cPn216 Chelate

For details of the synthesis of technetium chelate cPn216 the reader isreferred to patent filing GB0116815.2

1 b) Synthesis of cPn216-Glutaric Acid Intermediate

cPn216 (100 mg, 0.29 mmol) was dissolved in DMF (10 mL) and glutaricanhydride (33 mg, 0.29 mmol) added by portions with stirring. Thereaction was stirred for 23 hours to afford complete conversion to thedesired product. The pure acid was obtained following RP-HPLC in goodyield.

1 c) Synthesis of tetrafluorothiophenyl ester of cPn216-glutaric acid

To cPn216-glutaric acid (300 mg, 0.66 mmol) in DMF (2 mL) was added HATU(249 mg, 0.66 mmol) and NMM (132 μL, 1.32 mmol). The mixture was stirredfor 5 minutes then tetrafluorothiophenol (0.66 mmol, 119 mg) was added.The solution was stirred for 10 minutes then the reaction mixture wasdiluted with 20% acetonitrile/water (8 mL) and the product purified byRP-HPLC yielding 110 mg of the desired product following freeze-drying.

1 d) Synthesis of ClCH₂CO-Lys-Cys(tBu)-Arg-Gly-Asp-Cys(tBu)-Phe-Cys-NH₂(SEQ ID NO. 1)

The peptide was synthesised on an ABI 433A automatic peptide synthesiserstarting with Rink Amide AM resin on a 0.25 mmol scale using 1 mmolamino acid cartridges. The amino acids were pre-activated using HBTUbefore coupling. N-terminal amine groups were chloroacetylated using asolution of chloroacetic anhydride in DMF for 30 min.

The simultaneous removal of peptide and side-chain protecting groups(except tBu) from the resin was carried out in TFA containing TIS (5%),H₂0 (5%) and phenol (2.5%) for two hours.

After work-up 295 mg of crude peptide was obtained (Analytical HPLC:Gradient, 5-50% B over 10 min where A=H₂O/0.1% TFA and B=CH₃CN/0.1% TFA;column, Phenomenex Luna 3μ C18 (2) 50×4.6 mm; flow, 2 mL/min; detection,UV 214 nm; product retention time, 6.42 min). Further productcharacterisation was carried out using mass spectrometry: Expected, M+Hat 1118.5, found, at 1118.6).

1 e) Synthesis of thioethercyclo[CH₂CO-Lys-Cys(tBu)-Arg-Gly-Asp-Cys(tBu)-Phe-Cys]-NH₂ (SEQ ID NO.1)

295 mg of ClCH₂CO-Lys-Cys(tBu)-Arg-Gly-Asp-Cys(tBu)-Phe-Cys-NH₂ (SEQ IDNO. 1) was dissolved in water/acetonitrile. The mixture was adjusted topH 8 with ammonia solution and stirred for 16 hours.

After work-up 217 mg of crude peptide was obtained (Analytical HPLC:Gradient, 5-50% B over 10 min where A=H₂O/0.1% TFA and B=CH₃CN/0.1% TFA;column, Phenomenex Luna 3μ C18 (2) 50×4.6 mm; flow, 2 mL/min; detection,UV 214 nm; product retention time, 6.18 min). Further productcharacterisation was carried out using mass spectrometry: Expected, M+Hat 1882.5, found, at 1882.6).

1 f) Synthesis of Disulphide [Cys²⁻⁶] thioethercyclo[CH₂CO-Lys-Cys²-Arg-Gly-Asp-Cys⁶-Phe-Cys]-NH₂ (SEQ ID NO. 1)

217 mg of thioethercyclo[CH₂CO-Lys-Cys(tBu)-Arg-Gly-Asp-Cys(tBu)-Phe-Cys]-NH₂ (SEQ IDNO. 1) was treated with a solution of anisole (500 μL), DMSO (2 mL) andTFA (100 mL) for 60 min following which the TFA was removed in vacuo andthe peptide precipitated by the addition of diethyl ether.

Purification by preparative HPLC (Phenomenex Luna 10μ C18 (2) 250×50 mmcolumn) of the crude material (202 mg) was carried out using 0-30% B,where A=H₂O/0.1% TFA and B=CH₃CN/0.1% TFA, over 60 min at a flow rate of50 mL/min. After lyophilisation 112 mg of pure material was obtained(Analytical HPLC: Gradient, 5-50% B over 10 min where A=H₂O/0.1% TFA andB=CH₃CN/0.1% TFA; column, Phenomenex Luna 3μ C18 (2) 50×4.6 mm; flow, 2mL/min; detection, UV 214 nm; product retention time, 5.50 min). Furtherproduct characterisation was carried out using mass spectrometry:Expected, M+H at 968, found, at 971).

1 g) Synthesis of disulfide [Cys²⁻⁶] thioethercyclo[CH₂CO-Lys(cPn216-glutaryl)-Cys²-Arg-Gly-Asp-Cys⁶-Phe-Cys]-NH₂ (SEQID NO. 1)

9.7 mg of disulphide[Cys²⁻⁶] thioethercyclo[CH₂CO-Lys-Cys-Arg-Gly-Asp-Cys-Phe-Cys]-NH₂, (SEQ ID NO. 1) 9.1 mgof cPn216 chelate active ester and 6 μL of N-methylmorpholine wasdissolved in DMF (0.5 mL). The mixture was stirred for 3 hours.

Purification by preparative HPLC (Phenomenex Luna 5μ C18 (2) 250×21.20mm column) of the reaction mixture was carried out using 0-30% B, whereA=H₂O/0.1% TFA and B=CH₃CN/0.1% TFA, over 40 min at a flow rate of 10mL/min. After lyophilisation 5.7 mg of pure material was obtained(Analytical HPLC: Gradient, 0-30% B over 10 min where A=H₂O/0.1% TFA andB=CH₃CN/0.1% TFA; column, Phenomenex Luna 3μ C18 (2) 50×4.6 mm; flow, 2mL/min; detection, UV 214 nm; product retention time, 7.32 min). Furtherproduct characterisation was carried out using mass spectrometry:Expected, M+H at 1407.7, found, at 1407.6).

Example 2 Synthesis of disulphide [Cys²⁻⁶] thioethercyclo[CH₂CO-Lys(cPn216-glutaryl)-Cys²-Arg-Gly-Asp-Cys⁶-Phe-Cys]-(PEG)n-NH₂where n=1. (SEQ ID NO. 1)

2 a) Synthesis of17-(Fmoc-amino)-5-oxo-6-aza-3,9,12,15-tetraoxaheptadecanoic acid

This building block is coupled to the solid-phase using Fmoc chemistry.The coupled form of this building block will be referred to in short as(PEG)_(n) where n is a positive integer.

1,11-Diazido-3,6,9-trioxaundecane

A solution of dry tetraethylene glycol (19.4 g, 0.100 mol) andmethanesulphonyl chloride (25.2 g, 0.220 mol) in dry THF (100 ml) waskept under argon and cooled to 0° C. in an ice/water bath. To the flaskwas added a solution of triethylamine (22.6 g, 0.220 mol) in dry THF (25ml) dropwise over 45 min. After 1 hr the cooling bath was removed andstirring was continued for 4 hrs. Water (60 ml) was added. To themixture was added sodium hydrogencarbonate (6 g, to pH 8) and sodiumazide (14.3 g, 0.220 mmol), in that order. THF was removed bydistillation and the aqueous solution was refluxed for 24 h (two layersformed). The mixture was cooled and ether (100 ml) was added. Theaqueous phase was saturated with sodium chloride. The phases wereseparated and the aqueous phase was extracted with ether (4×50 ml).Combined organic phases were washed with brine (2×50 ml) and dried(MgSO₄). Filtration and concentration gave 22.1 g (91%) of yellow oil.The product was used in the next step without further purification.

11-Azido-3,6,9-trioxaundecanamine

To a mechanically, vigorously stirred suspension of1,11-diazido-3,6,9-trioxaundecane (20.8 g, 0.085 mol) in 5% hydrochloricacid (200 ml) was added a solution of triphenylphosphine (19.9 g, 0.073mol) in ether (150 ml) over 3 hrs at room temperature. The reactionmixture was stirred for additional 24 hrs. The phases were separated andthe aqueous phase was extracted with dichloromethane (3×40 ml). Theaqueous phase was cooled in an ice/water bath and pH was adjusted to ca12 by addition of KOH. The product was extracted into dichloromethane(5×50 ml). Combined organic phases were dried (MgSO₄). Filtration andevaporation gave 14.0 g (88%) of yellow oil. Analysis by MALDI-TOF massspectroscopy (matrix: α-cyano-4-hydroxycinnamic acid) gave a M+H peak at219 as expected. Further characterisation using ¹H (500 MHz) and ¹³C(125 MHz) NMR spectroscopy verified the structure.

17-Azido-5-oxo-6-aza-3,9,12,15-tetraoxaheptadecanoic acid

To a solution of 11-azido-3,6,9-trioxaundecanamine (10.9 g, 50.0 mmol)in dichloromethane (100 ml) was added diglycolic anhydride (6.38 g, 55.0mmol). The reaction mixture was stirred overnight. HPLC analysis (columnVydac 218TP54; solvents: A=water/0.1% TFA and B=acetonitrile/0.1% TFA;gradient 4-16% B over 20 min; flow 1.0 ml/min; UV detection at 214 and284 nm), showed complete conversion of starting material to a productwith retention time 18.3 min. The solution was concentrated to givequantitative yield of a yellow syrup. The product was analysed by LC-MS(ES ionisation) giving [MH]+ at 335 as expected. ¹H (500 MHz) and ¹³C(125 MHz) NMR spectroscopy was in agreement with structure The productwas used in the next step without further purification.

17-Amino-5-oxo-6-aza-3,9,12,15-tetraoxaheptadecanoic acid

A solution of 17-azido-5-oxo-6-aza-3,9,12,15-tetraoxaheptadecanoic acid(8.36 g, 25.0 mmol) in water (100 ml) was reduced using H₂(g)-Pd/C(10%). The reaction was run until LC-MS analysis showed completeconversion of starting material (column Vydac 218TP54; solvents:A=water/0.1% TFA and B=acetonitrile/0.1% TFA; gradient 4-16% B over 20min; flow 1.0 ml/min; UV detection at 214 and 284 nm, ES ionisationgiving M+H at 335 for starting material and 309 for the product). Thesolution was filtered and used directly in the next step.

17-(Fmoc-amino)-5-oxo-6-aza-3,9,12,15-tetraoxaheptadecanoic acid

To the aqueous solution of17-amino-5-oxo-6-aza-3,9,12,15-tetraoxaheptadecanoic acid from above(corresponding to 25.0 mmol amino acid) was added sodium bicarbonate(5.04 g, 60.0 mmol) and dioxan (40 ml). A solution of Fmoc-chloride(7.11 g, 0.275 mol) in dioxan (40 ml) was added dropwise. The reactionmixture was stirred overnight. Dioxan was evaporated off (rotavapor) andthe aqueous phase was extracted with ethyl acetate. The aqueous phasewas acidified by addition of hydrochloric acid and precipitated materialwas extracted into chloroform. The organic phase was dried (MgSO₄),filtered and concentrated to give 11.3 g (85%) of a yellow syrup. Thestructure was confirmed by LC-MS analysis (column Vydac 218TP54;solvents: A=water/0.1% TFA and B=acetonitrile/0.1% TFA; gradient 40-60%B over 20 min; flow 1.0 ml/min; UV detection at 214 and 254 nm, ESionisation giving M+H at 531 as expected for the product peak at 5.8minutes). The analysis showed very low content of side products and thematerial was used without further purification.

2 b) Synthesis ofClCH₂CO-Lys-Cys(tBu)-Arg-Gly-Asp-Cys(tBu)-Phe-Cys-(PEG)n-NH₂ where n=1(SEQ ID NO. 1)

The PEG unit was coupled manually to Rink Amide AM resin, starting on a0.25 mmol scale, mediated by HATU activation. The remaining peptide wasassembled on an ABI 433A automatic peptide synthesiser using 1 mmolamino acid cartridges. The amino acids were pre-activated using HBTUbefore coupling. N-terminal amine groups were chloroacetylated using asolution of chloroacetic anhydride in DMF for 30 min.

The simultaneous removal of peptide and side-chain protecting groups(except tBu) from the resin was carried out in TFA containing TIS (5%),H₂0 (5%) and phenol (2.5%) for two hours.

After work-up 322 mg of crude peptide was obtained (Analytical HPLC:Gradient, 5-50% B over 10 min where A=H₂O/0.1% TFA and B=CH₃CN/0.1% TFA;column, Phenomenex Luna 3μ C18 (2) 50×4.6 mm; flow, 2 mL/min; detection,UV 214 nm; product retention time, 6.37 min). Further productcharacterisation was carried out using mass spectrometry: Expected, M+Hat 1409, found, at 1415).

2 c) Synthesis of thioethercyclo[CH₂CO-Lys-Cys(tBu)-Arg-Gly-Asp-Cys(tBu)-Phe-Cys]-(PEG)n-NH₂ wheren=1 (SEQ ID NO. 1)

322 mg of ClCH₂CO-Lys-Cys(tBu)-Arg-Gly-Asp-Cys(tBu)-Phe-Cys-(PEG)n-NH₂(SEQ ID NO. 1) was dissolved in water/acetonitrile. The mixture wasadjusted to pH 8 with ammonia solution and stirred for 16 hours.

After work-up crude peptide was obtained (Analytical HPLC: Gradient,5-50% B over 10 min where A=H₂O/0.1% TFA and B=CH₃CN/0.1% TFA; column,Phenomenex Luna 3μ C18 (2) 50×4.6 mm; flow, 2 mL/min; detection, UV 214nm; product retention time, 6.22 min). Further product characterisationwas carried out using mass spectrometry: Expected, M+H at 1373, found,at 1378).

2 d) Synthesis of disulphide [Cys²⁻⁶] thioethercyclo[CH₂CO-Lys-Cys²-Arg-Gly-Asp-Cys⁶-Phe-Cys]-(PEG)n-NH₂ where n=1 (SEQID NO. 1)

Thioethercyclo[CH₂CO-Lys-Cys(tBu)-Arg-Gly-Asp-Cys(tBu)-Phe-Cys]-(PEG)n-NH₂ (SEQID NO. 1) was treated with a solution of anisole (200 μL), DMSO (2 mL)and TFA (100 mL) for 60 min following which the TFA was removed in vacuoand the peptide precipitated by the addition of diethyl ether.

Purification by preparative HPLC (Phenomenex Luna 5μ C18 (2) 250×21.20mm column) of 70 mg crude material was carried out using 0-30% B, whereA=H₂O/0.1% TFA and B=CH₃CN/0.1% TFA, over 40 min at a flow rate of 10mL/min. After lyophilisation 46 mg of pure material was obtained(Analytical HPLC: Gradient, 0-30% B over 10 min where A=H₂O/0.1% TFA andB=CH₃CN/0.1% TFA; column, Phenomenex Luna 3μ C18 (2) 50×4.6 mm; flow, 2mL/min; detection, UV 214 nm; product retention time, 6.80 min). Furtherproduct characterisation was carried out using mass spectrometry:Expected, M+H at 1258.5, found, at 1258.8).

2 e) Synthesis of disulfide [Cys²⁻⁶] thioethercyclo[CH₂CO-Lys(cPn216-glutaryl)-Cys²-Arg-Gly-Asp-Cys⁶-Phe-Cys]-(PEG)n-NH₂where n=1 (SEQ ID NO. 1)

13 mg of [Cys²⁻⁶]cyclo[CH₂CO-Lys-Cys-Arg-Gly-Asp-Cys-Phe-Cys]-(PEG)n-NH₂, (SEQ ID NO. 1)9.6 mg of cPn216 chelate active ester and 8 μL of N-methylmorpholine wasdissolved in DMF (0.5 mL). The mixture was stirred for 2 hours and 30minutes.

Purification by preparative HPLC (Phenomenex Luna 5μ C18 (2) 250×21.20mm column) of the reaction mixture was carried out using 0-30% B, whereA=H₂O/0.1% TFA and B=CH₃CN/0.1% TFA, over 40 min at a flow rate of 10mL/min. After lyophilisation 14.2 mg of pure material was obtained(Analytical HPLC: Gradient, 0-30% B over 10 min where A=H₂O/0.1% TFA andB=CH₃CN/0.1% TFA; column, Phenomenex Luna 3μ C18 (2) 50×4.6 mm; flow, 2mL/min; detection, UV 214 nm; product retention time, 7.87 min). Furtherproduct characterisation was carried out using mass spectrometry:Expected, M+H at 1697.8, found, at 1697.9).

Example 3 Synthesis of disulfide [Cys²⁻⁶] thioethercyclo[CH₂CO-Lys(cPn216-glutaryl)-Cys²-Arg-Gly-Asp-Cys⁶-Phe-Cys]-(PEG)n-NH₂where n=2. (SEQ ID NO. 1)

3 a) Synthesis ofClCH₂CO-Lys-Cys(tBu)-Arg-Gly-Asp-Cys(tBu)-Phe-Cys-(PEG)n-NH₂ where n=2(SEQ ID NO. 1)

Assembly of peptide as for example 2 b), both PEG units coupledmanually.

After work-up crude peptide was obtained (Analytical HPLC: Gradient,5-50% B over 10 min where A=H₂O/0.1% TFA and B=CH₃CN/0.1% TFA; column,Phenomenex Luna 3μ C18 (2) 50×4.6 mm; flow, 2 mL/min; detection, UV 214nm; product retention time, 6.40 min).

3 b) Synthesis of thioethercyclo[CH₂CO-Lys-Cys(tBu)-Arg-Gly-Asp-Cys(tBu)-Phe-Cys]-(PEG)n-NH₂ wheren=2 (SEQ ID NO. 1)

ClCH₂CO-Lys-Cys(tBu)-Arg-Gly-Asp-Cys(tBu)-Phe-Cys-(PEG)n-NH₂ (SEQ IDNO. 1) where n=2 was dissolved in water/acetonitrile. The mixture wasadjusted to pH 8 with ammonia solution and stirred for 16 hours.

After work-up 380 mg of crude peptide was obtained (Analytical HPLC:Gradient, 5-50% B over 10 min where A=H₂O/0.1% TFA and B=CH₃CN/0.1% TFA;column, Phenomenex Luna 3μ C18 (2) 50×4.6 mm; flow, 2 mL/min; detection,UV 214 nm; product retention time, 6.28 min). Further productcharacterisation was carried out using mass spectrometry: Expected, M+Hat 1663, found, at 1670).

3 c) Synthesis of disulphide [Cys²⁻⁶] thioethercyclo[CH₂CO-Lys-Cys²-Arg-Gly-Asp-Cys⁶-Phe-Cys]-(PEG)n-NH₂ where n=2.(SEQ ID NO. 1)

380 mg of thioethercyclo[CH₂CO-Lys-Cys(tBu)-Arg-Gly-Asp-Cys(tBu)-Phe-Cys]-(PEG)n-NH₂ wheren=2 (SEQ ID NO. 1) was treated with a solution of anisole (500 μL), DMSO(2 mL) and TFA (100 mL) for 60 min following which the TFA was removedin vacuo and the peptide precipitated by the addition of diethyl ether.

Purification by preparative HPLC (Phenomenex Luna 10μ C18 (2) 250×50 mmcolumn) of the crude material (345 mg) was carried out using 0-30% B,where A=H₂O/0.1% TFA and B=CH₃CN/0.1% TFA, over 60 min at a flow rate of50 mL/min. After lyophilisation 146 mg of pure material was obtained(Analytical HPLC: Gradient, 0-30% B over 10 min where A=H₂O/0.1% TFA andB=CH₃CN/0.1% TFA; column, Phenomenex Luna 3μ C18 (2) 50×4.6 mm; flow, 2mL/min; detection, UV 214 nm; product retention time, 7.42 min). Furtherproduct characterisation was carried out using mass spectrometry:Expected, M+H at 1548.6, found, at 1548.8).

3 d) Synthesis of disulphide [Cys²⁻⁶] thioethercyclo[CH₂CO-Lys(cPn216-glutaryl)-Cys²-Arg-Gly-Asp-Cys⁶-Phe-Cys]-(PEG)n-NH₂where n=2. (SEQ ID NO. 1)

146 mg of [Cys²⁻⁶]cyclo[CH₂CO-Lys-Cys-Arg-Gly-Asp-Cys-Phe-Cys]-(PEG)₂-NH₂, (SEQ ID NO. 1)110 mg of cPn216 chelate active ester and 76 μL of N-methylmorpholinewas dissolved in DMF (6 mL). The mixture was stirred for 9 hours.

Purification by preparative HPLC (Phenomenex Luna 10μ C18 (2) 250×50 mmcolumn) of the reaction mixture was carried out using 0-30% B, whereA=H₂O/0.1% TFA and B=CH₃CN/0.1% TFA, over 60 min at a flow rate of 50mL/min. After lyophilisation 164 mg of pure material was obtained(Analytical HPLC: Gradient, 0-30% B over 10 min where A=H₂O/0.1% TFA andB=CH₃CN/0.1% TFA; column, Phenomenex Luna 3μ C18 (2) 50×4.6 mm; flow, 2mL/min; detection, UV 214 nm; product retention time, 8.13 min). Furtherproduct characterisation was carried out using mass spectrometry:Expected, M+H at 1988.0, found, at 1988.0).

Example 4 Synthesis of disulfide [Cys²⁻⁶] thioethercyclo[CH₂CO-Lys(cPn216-glutaryl)-Cys²-Arg-Gly-Asp-Cys⁶-Phe-Cys]-(PEG)n-NH₂where n=4. (SEQ ID NO. 1)

4 a) Synthesis ofClCH₂CO-Lys-Cys(tBu)-Arg-Gly-Asp-Cys(tBu)-Phe-Cys-(PEG)n-NH₂ where n=4(SEQ ID NO. 1)

Assembly of peptide as for example 2 b), all four PEG units coupledmanually.

After work-up crude peptide was obtained (Analytical HPLC: Gradient,5-50% B over 10 min where A=H₂O/0.1% TFA and B=CH₃CN/0.1% TFA; column,Phenomenex Luna 3μ C18 (2) 50×4.6 mm; flow, 2 mL/min; detection, UV 214nm; product retention time, 6.50 min).

4 b) Synthesis of thioethercyclo[CH₂CO-Lys-Cys(tBu)-Arg-Gly-Asp-Cys(tBu)-Phe-Cys]-(PEG)n-NH₂ wheren=4 (SEQ ID NO. 1)

ClCH₂CO-Lys-Cys(tBu)-Arg-Gly-Asp-Cys(tBu)-Phe-Cys-(PEG)₄-NH₂ (SEQ IDNO. 1) was dissolved in water/acetonitrile. The mixture was adjusted topH 8 with ammonia solution and stirred for 16 hours.

After work-up crude peptide was obtained (Analytical HPLC: Gradient,5-50% B over 10 min where A=H₂O/0.1% TFA and B=CH₃CN/0.1% TFA; column,Phenomenex Luna 3μ C18 (2) 50×4.6 mm; flow, 2 mL/min; detection, UV 214nm; product retention time, 6.37 min). Further product characterisationwas carried out using mass spectrometry: Expected, [(M+2H)/2] at 1122.0,found, at 1122.5).

4 c) Synthesis of disulphide [Cys²⁻⁶] thioethercyclo[CH₂CO-Lys-Cys²-Arg-Gly-Asp-Cys⁶-Phe-Cys]-(PEG)n-NH₂ where n=4 (SEQID NO. 1)

Thioethercyclo[CH₂CO-Lys-Cys(tBu)-Arg-Gly-Asp-Cys(tBu)-Phe-Cys]-(PEG)₄-NH₂ (SEQID NO. 1) was treated with a solution of anisole (100 μL), DMSO (1 mL)and TFA (50 mL) for 60 min following which the TFA was removed in vacuoand the peptide precipitated by the addition of diethyl ether.

Purification by preparative HPLC (Phenomenex Luna 5μ C18 (2) 250×21.20mm column) of the crude material (345 mg) was carried out using 5-50% B,where A=H₂O/0.1% TFA and B=CH₃CN/0.1% TFA, over 40 min at a flow rate of10 mL/min. After lyophilisation 12 mg of pure material was obtained(Analytical HPLC: Gradient, 5-50% B over 10 min where A=H₂O/0.1% TFA andB=CH₃CN/0.1% TFA; column, Phenomenex Luna 3μ C18 (2) 50×4.6 mm; flow, 2mL/min; detection, UV 214 nm; product retention time, 4.87 min).

4 d) Synthesis of disulphide [Cys²⁻⁶] thioethercyclo[CH₂CO-Lys(cPn216-glutaryl)-Cys²-Arg-Gly-Asp-Cys⁶-Phe-Cys]-(PEG)n-NH₂where n=4. (SEQ ID NO. 1)

12 mg of disulphide [Cys²⁻⁶] thioethercyclo[CH₂CO-Lys-Cys-Arg-Gly-Asp-Cys-Phe-Cys]-(PEG)₄-NH₂, (SEQ ID NO. 1)5.2 mg of cPn216 chelate active ester and 2 μL of N-methylmorpholine wasdissolved in DMF (0.5 mL). The mixture was stirred for 7 hours.

Purification by preparative HPLC (Phenomenex Luna 5μ C18 (2) 250×21.20mm column) of the reaction mixture was carried out using 5-50% B, whereA=H₂O/0.1% TFA and B=CH₃CN/0.1% TFA, over 40 min at a flow rate of 10mL/min. After lyophilisation 8 mg of pure material was obtained(Analytical HPLC: Gradient, 5-50% B over 10 min where A=H₂O/0.1% TFA andB=CH₃CN/0.1% TFA; column, Phenomenex Luna 3μ C18 (2) 50×4.6 mm; flow, 2mL/min; detection, UV 214 nm; product retention time, 5.17 min). Furtherproduct characterisation was carried out using mass spectrometry:Expected, [(M+2H)/2] at 1284.6, found, at 1284.9).

1. A pharmaceutical composition comprising an effective amount of acompound of formula (I) or a pharmaceutically acceptable salt thereof,together with one or more pharmaceutically acceptable adjuvants,excipients or diluents:

wherein: G represents glycine D represents aspartic acid R₁ represents—(CH₂)_(n)— or —(CH₂)_(n)—C₆H₄— wherein n represents a positive integer1 to 10 h represents a positive integer 1 or 2 X₁ represents an aminoacid residue wherein said amino acid possesses a functional side-chainsuch as an acid or amine, X₂ and X₄ represent independently an aminoacid residue capable of forming a disulfide bond, X₃ representsarginine, N-methylarginine or an arginine mimetic, X₅ represents ahydrophobic amino acid or derivatives thereof, and X₆ represents athiol-containing amino acid residue, and X₇ is absent or represents abiomodifier moiety, Z₁ comprises a chelating agent and/or a radionuclidereporter moiety and W₁ is absent or represents a spacer moiety.
 2. Thecomposition of claim 1, wherein any of the amino acid residues areindependently in the D or L conformation.
 3. The composition of claim 1,wherein R₁ represents —(CH₂)—.
 4. The composition of claim 1, wherein X₁represents aspartic acid, glutamic acid, lysine, homolysine or adiaminoalkylic acid or derivatives thereof.
 5. The composition of claim1, wherein X₂, X₄ and X₆ independently represent a cysteine orhomocysteine residue.
 6. The composition of claim 1, wherein X₃represents an arginine residue.
 7. The composition of claim 1, whereinX₅ represents a tyrosine, a phenylalanine, a 3-iodo-tyrosine or anaphthylalanine residue.
 8. The composition of claim 1, wherein X₇ isabsent or comprises 1-10 units of a monodisperse PEG building block. 9.The composition of claim 1, wherein X₇ is absent or comprises 1-10 unitsof Formula II


10. The composition of claim 1, wherein X₇ represent 1-10 amino acidresidues.
 11. The composition of claim 1, wherein X₇ represent glycine,lysine, aspartic acid or serine residues.
 12. The composition of claim1, where Z₁ comprises a chelating agent of Formula III

where: each R¹, R², R³ and R⁴ is independently an R group; each R groupis independently H or C₁₋₁₀ alkyl, C₃₋₁₀ alkylaryl, C₂₋₁₀ alkoxyalkyl,C₁₋₁₀ hydroxyalkyl, C₁₋₁₀ alkylamine, C₁₋₁₀ fluoroalkyl, or 2 or more Rgroups, together with the atoms to which they are attached form acarbocyclic, heterocyclic, saturated or unsaturated ring.
 13. Thecomposition of claim 1, where Z₁ comprises a chelating agent of formula:


14. The composition of claim 1, wherein Z₁ comprises a radionuclidereporter moiety, wherein when said radionuclide is a radiometal, saidradiometal is chelated by the chelating agent of Z₁.
 15. The compositionof claim 14, wherein the reporter moiety comprises ⁹⁰Y, ^(99m)Tc, ¹¹¹In,⁴⁷Sc, ⁶⁷Ga, ⁵¹Cr, ^(177m)Sn, ⁶⁷Cu, ¹⁶⁷Tm, ⁹⁷Ru, ¹⁸⁸Re, ¹⁷⁷Lu, ¹⁹⁹Au,²⁰³Pb, ¹⁴¹Ce or ¹⁸F.
 16. The composition of claim 15, wherein theradionuclide reporter moiety is ^(99m)Tc.
 17. The composition of claim1, where W₁ is glutaric or succinic acid.
 18. The composition of claim1, wherein the compound of Formula (I) is defined by the followingformulae:


19. A method of generating images of a human or animal body involvingadministering a contrast agent to said body, and generating an image ofat least a part of said body to which said contrast agent hasdistributed, wherein said contrast agent comprises the composition ofclaim
 14. 20. The method of claim 19, which is carried out to monitorthe effect of treatment of a human or animal body with a drug to combata condition associated with cancer, wherein said administration andimage generation are optionally but preferably being effectedrepeatedly, e.g. before, during and after treatment with said drug.